J. Membrane Biol. 38, 51-72 (1978)

Effects of Calcium Ionophores on the Transport and Distribution of Calcium in Isolated Cells and in Liver and Kidney Slices Andr6 B. Borle and Rebecca Studer Department of Physiology,University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 Received 15 February 1977; revised 18 May 1977

Summary. The effects of calcium ionophores on cellular calcium metabolism were studied in cultured kidney cells, in cells freshly isolated from rat kidney, and in liver and kidney slices. In isolated cells, these ionophores decreased the total cellular Ca content and the mitochondrial Ca. 4~Ca efflux from prelabelled cells was also stimulated even in the absence of extracellular Ca. In slices, the ionophore A23187 increased the total slice Ca and the uptake of ~SCa. However, the mitochondria isolated from these slices treated with the ionophore had a lower total Ca and a depressed relative radioactivity. These results suggest that the increased cytosolic Ca produced by Ca ionophores may be due to mobilization of intracellular Ca stores rather than to a net shift of Ca from the extracellular fluids to the cell.

The lipophilic antibiotics A23187 and X-537A are known to increase the permeability of biological membranes to divalent cations (Reed & Lardy, 1972a; Pressman, 1973). These ionophores are frequently used as a tool to study the role of Ca in several cellular functions. All the results published to date support the view that Ca ionophores increase the concentration of free Ca in the cell cytosol. The source of Ca responsible for the rise in cytosolic Ca is usually assumed to be extracellular. However, the contribution of any Ca sequestered in subcellular structures to the rise in cytosolic Ca is unclear. It is known that ionophores trigger the release of Ca from isolated mitochondria (Reed & Lardy, 1972b; Sordahl, 1974; Binet & Volfin, 1975). Furthermore, it has been observed that Ca ionophores increase cytosolic Ca in some tissues even in the absence of Ca in the extracellular fluids (Binet & Volfin, 1975; Chambers, Pressman & Rose, 1974). It is therefore possible that the Ca sequestered in subcellular structures may contribute significantly to the rise in cytosolic Ca produced by Ca ionophores. 022-2631/78/0038-0051 $4.40 9 Springer-Verlag New York Inc. 1978

52

A.B. Borle and R. Studer

It has also been reported that the Ca ionophores A23187 and X-537A increase incorporation of 45Ca by some cells and tissues (Reed & Lardy, 1972a; Prince, Rasmussen & Berridge, 1973; Massini & Luscher, 1974; Diziak & Stern, 1975). However, some investigators have demonstrated that the same ionophores decrease the cell Ca concentration in other tissues (Holland, Armstrong & Steinberg, 1975; Schudt & Pette, 1975). These ionophores could possibly increase the total cell Ca in some tissues and decrease it in others; however, the possibility of artifacts should not be excluded. Indeed one cannot exlude that an increased 45Ca incorporation by a tissue may coexist with a decreased cellular Ca. This study reports the effects of Ca ionophores in cultured kidney cells, in cells freshly isolated from rat kidney, and in rat liver and kidney slices. We found that in isolated cells these ionophores decrease the cellular Ca content and deplete the mitochondria of a significant fraction of their Ca. In kidney and liver slices, on the other hand, A23187 increased 4SCa uptake and the total Ca concentration of the tissue. However, the mitochondria isolated from slices which had gained Ca and had an increased 45Ca were found to have a lower total Ca and a decreased relative radioactivity after the addition of A23187.

Materials and Methods Red blood cells. Human red blood cells were obtained by venipuncture, washed twice in Krebs-Henseleit buffer and used in suspension at a concentration of 1 to 2 mg cell protein/ml of medium. Cultured cells. Monkey kidney cells (LLC-MK2) were grown as monolayers in minimum essential medium (MEM) and Earl's salt solutions. They were harvested after one week and incubated as a suspension in a Krebs-Henseleit buffer for the experiments. Isolated renal cells and tubules. Renal cells and tubules were prepared by the method published by Shain (1972). The buffer used was modified slightly as follows (buffer z): 20 mM NaHEPES, pH 7.4; 120 mM NaC1; 1 m~ MgS04 ; 3 mM K2HPO~; 1 mM CaCI2; 1 mg/ml bovine serum albumin fraction V ; 10,000 units potassium penicillin G and 10,000 lag streptomycin sulfate/100 ml (Microbiological Associates, Bethesda, Md.) ; 0.4 mg/ml of collagenase (Worthington Biochemical Corp., Freehold, N.J.). Sprague-Dawley rats, 100-150 g, were decapitated and bled. The abdominal cavity was opened and the aorta ligated proximal to the renal artery. A blunt needle was introduced into the abdominal aorta distal to the renal arteries and the kidneys were perfused with 40 ml of saline at 37 ~ to remove the blood remaining in this vascular bed and then with 10 ml of the buffer containing collagenase. The kidneys were removed, decapsulated, minced with scissors, and incubated for 1 hr at 37 ~ in buffer z. The tissue mince was dispersed by pipetting the suspension several times every 15 min. The suspension was filtered through a 110 lam sieve and the

Ca Ionophores on Cellular Calcium

53

cells thoroughly washed with buffer z. The cells were centrifuged at 150 x g, the suspending medium containing the collagenase was discarded, and the cells were washed once with the Krebs Heinseleit buffer to be used for the experiments. The cells were preincubated for 1 hr before the start of the experiments. Under phase contrast microscopy, the suspension consisted of isolated cells, clumps of cells, and of very small fragments of tubules.

Slices. The liver and the kidneys of 100 150 g Sprague Dawley rats were removed, after decapitation, and placed in an ice cold Krebs buffer. Slices, 0.5-mm thick, were prepared in a cold room with a Mickel microtome. The slices were incubated in Krebs Henseleit bicarbonate buffer. Cell-fractionation. The cells were homogenized at 0~ with a Bellco glass tissue grinder in 250 mM sucrose containing 0.1 mM EGTA to prevent redistribution of Ca during the fractionation process. The homogenate was centrifuged for 20 min at 800 x g and the supernatant centrifuged twice at 18,000 x g. The sucrose in which the mitochondria were suspended during the second centrifugation contained no EGTA. Slices mitochondria. Liver and kidney slices were prepared and incubated as previously described. The slices were labeled with ~SCa for 2 hr as for a determination of Ca uptake. After 2 hr, A23187 was added to the incubation medium of the experimental group at a concentration of 5 gg/ml. Control groups received ethanol at a final concentration of 0.5%. Ten rain after the addition of the ionophore or of ethanol, the slices were collected and washed as previously described. They were homogenized in a cold room in a 12-ml Potter-Elvehjem grinder with a Teflon pestle, in 250 mM sucrose containing 0.1 mM EGTA to prevent Ca redistribution among subcellular components. The homogenate was centrifuged for 20 rain at 800 x g and the supernatant centrifuged at 18,000 x g on a Beckman L-100 ultracentrifuge. The mitochondrial pellet was washed in sucrose devoid of EGTA, resuspended and recentrifuged at 18,000xg in sucrose without EGTA. The final pellet was homogenized with an ultrasonic probe and its total Ca concentration, its radioactivity, and its protein concentration were determined. Incubating medium. The medium of incubation was a Krebs Henseleit bicarbonate buffer containing (in raM): 140 Na +, 5 K +, 24 HCO~, 121 CI-, 1 MgSO4, 1 CaCI2 and 1 Na2HPO~: NaH2PO4 at pH 7.4. The gas phase consisted of 95% air, 5% CO2. Ionophores. The ionophores A23187 and X-537Awere dissolved in ethanol. A23187 was added to the cell suspension at concentrations from 0.01 to 5 gg/ml. The concentration of ethanol in the medium never exceeded 0.1%. A few experiments were performed with X-537A at a concentration of 10 ~tg/ml. Isolated cell calcium uptake. 1.5 to 2.0 ml of cells centrifuged at 150 • g were suspended in 60 ml of medium with a gas phase of 5% CO2 in air. 45Ca was added after 2 hr of preincubation and uptake determined by methods previously published (Borle, 1975). Isolated cells calcium eflTux. 0.6 ml of cells centrifuged at 150 x g were first preincubated for 1 hr, then labeled with 45Ca for 60 rain. Isotopic desaturations were performed and the efflux rate coefficient calculated according to the method previously published (Borle, 1975). Slices calcium uptake. After 30 rain of preincubation, the incubating medium of the slices was replaced with fresh buffer containing 100 ~tCi of 45Ca. The isotope uptake was determined for a control period of 2 hr and an experimental period of 2 hr. The slices

54

A.B. Borle and R. Studer

were removed with forceps from the incubating medium, and quickly washed in 4 different beakers containing a buffered saline solution kept at 0 ~ The slices were blotted lightly on filter paper and weighed on a Mettler balance. The uptake of isotope by the slices was determined at 5, l0 and 20 min after the addition of 45Ca and every 15 min thereafter. The ionophore was added at min 126 and tissue samples were taken at min 127, 137, 145, 155 and every 15 min thereafter, until rain 240. Each sample with a wet wt of 10-15 mg was placed in tared Pyrex vessel and dried for 24 hr at 95-100 ~ in a vacuum oven. The dry wt was determined and the samples were then ashed overnight in a muffle furnace at 500 ~ The ash was dissolved in 0.2 ml of 2 N HC1 and diluted to 2 ml with deionized water. The total Ca and the radioactivity were determined on the dissolved ash.

Definition of uptake. The uptake values of slices and cells were calculated by dividing the tissue (or cell) radioactivity by the medium specific activity. Uptake can also be called relative radioactivity and has the units of nmole/mg dry wt or nmole/mg protein: sample radioactivity (cpm/mg dry wet)/medium specific activity (cmp/nmole)=uptake (nmole/mg dry wt). Determinations. The cells were homogenized by an ultrasonic probe, their protein concentration measured by the Lowry method (Lowry et al., 1951). Ca was measured by fluorometric titration (Borle & Briggs, 1968). 45Ca was assayed by liquid scintillation spectrometry using Aquasol (New England Nuclear) on a Beckman L-100 counter.

Results

Red Blood Cells Calcium uptake by red blood cells. A23187 was a d d e d to a s u s p e n s i o n o f h u m a n e r y t h r o c y t e s at c o n c e n t r a t i o n s o f 1 a n d 5 gg/ml. M i c r o s c o p i c e x a m i n a t i o n o f the cells with p h a s e c o n t r a s t revealed t h a t 5 gg A 2 3 1 8 7 / m l c a u s e d the cells to swell a n d h e m o l y z e in less t h a n 5 rain. W i t h an i o n o p h o r e c o n c e n t r a t i o n o f 1 ~g/ml, h o w e v e r , there was n o a p p a r e n t h e m o l y s i s b u t the cells lost their b i c o n c a v e shape. Fig. 1 shows t h a t red b l o o d cells do n o t a c c u m u l a t e significant a m o u n t s o f tracer. 4~Ca u p t a k e is e x t r e m e l y small, 0.013 n m o l e / m g p r o t e i n ( T a b l e 1). W h e n 1 ~g A 2 3 1 8 7 / m l o f m e d i u m is a d d e d to the suspension, t h e r e is a n i m m e d i a t e u p t a k e o f isotope. Fig. 1 shows that the cells' relative r a d i o a c t i v i t y rises f r o m 0.013 to 3.1 n m o l e s / m g o f cell p r o t e i n , w i t h i n 10 rain. T h i r t y m i n u t e s after the a d d i t i o n o f the i o n o p h o r e , the r a d i o a c t i v i t y o f the cells declines. This m a y be due to a progressive hemolysis, because the p r o t e i n c o n c e n t r a t i o n o f the s u s p e n s i o n also declines in parallel fashion. T a b l e 1 shows that after the a d d i t i o n o f A23187 the t o t a l c a l c i u m c o n c e n t r a t i o n o f the e r y t h r o c y t e s increases 6 2 % , f r o m 9.62 to 15.6 n m o l e s / m g protein. H o w e v e r since the cell p r o t e i n c o n c e n t r a t i o n decreases 14%, the actual increase in C a m a y be less (36%). T h e rise in the total cell C a c o n c e n t r a -

Ca Ionophores on Cellular Calcium

55

"G-

"5 E ,._%= ",z"

4 5

EI -I--

o._

2

E o -6

905

o

OI 0

60

120

minutes Fig. 1. Effect of A23187 on Ca uptake (relative radioactivity) in human red blood cells. A23187 (1 gg/ml) was added at min 55 (closed circles). Control cells (open circles) received ethanol (0.1%) at min 55. The graph shows the means of 2 experiments in each group

Table 1. Effect of 1 gg A23187/ml on the total Ca concentration and on Ca uptake of human red blood cells ControI (n=t4)

A23187 (n=10)

%change

P

15.6 +_1.06

+62

<0.001

Cell calcium (nmole/mg protein)

9.62 +0.5

Calcium uptake (nmole/mg protein)

0.013_+0.002

2.99_+0.1

+22,900%

<0.001

Cell protein (mg/ml suspension)

1.41 +0.03

1.21_+0.05

-14%

<0.01

+36%

<0.01

+ 20,000%

< 0.001

Cell calcium (nmole/ml suspension) Calcium uptake (nmole/ml suspension)

13.4 -+0.6 0.018 + 0.003

18.2 -+ 1.41 3.62 + 0.1

Values are mean + SE.

tion is r e a s o n a b l y close to the value o f the C a u p t a k e m e a s u r e d with 45Ca. T h e s e e x p e r i m e n t s illustrate that, in cells d e v o i d o f m i t o c h o n d r i a , the i o n o p h o r e A23187 increases the p e r m e a b i l i t y o f the cell m e m b r a n e a n d allows a net shift o f C a f r o m the e x t r a c e l l u l a r fluids to the cell interior d o w n its t h e r m o d y n a m i c g r a d i e n t .

56

A.B. Borle and R. Studer

A A23187

6

e

0!i

6

-6

E

,rv

I,

4

.01

I

I

.I

I

5

A25187 pg/ml

E =( _ )

2 w

w

l

9

(3 s

0

I

I

I

I

2

:5

4

hours Fig. 2. (A): Effect of A23187 on Ca uptake (relative radioactivity) in kidney cells (LLCMK2). The cells were preincubated for 2 hr in a medium containing 1 m~ Ca and phosphate before the addition of ~SCa. A23t87 was added at min 55 at a concentration of t pg/ml. The values are the mean of 6 experiments. The standard errors of the means were smaller than 0.15 nmole/mg protein. (B): Cells' relative specific activity after A23187 addition as a function of the ionophore concentration

Cultured Monkey Kidney Cells Calcium uptake by kidney cells. T h e effect o f i o n o p h o r e s o n Ca u p t a k e by k i d n e y cells is m a r k e d l y different. If the i o n o p h o r e is a d d e d w h e n the cells are n e a r their i s o t o p i c equilibrium, the cell r a d i o a c t i v i t y d r o p s i m m e d i a t e l y . Fig. 2 shows the results o f 6 e x p e r i m e n t s o b t a i n e d with 1 gg A 2 3 1 8 7 / m l o f m e d i u m a n d Fig. 3 the results o f 4 e x p e r i m e n t s obt a i n e d with l0 pg X - 5 3 7 A / m l . S u c h a d r o p in r a d i o a c t i v i t y w h e n the cells are close to their i s o t o p i c e q u i l i b r i u m can only m e a n a net m o v e m e n t o f Ca o u t o f the cells. This is c o n f i r m e d b y the fact t h a t the total cell Ca d r o p s 3 0 % ( T a b l e 2).

5

Ca Ionophores on Cellular Calcium

0

57

X-557A

6

"6

4

2 #

II

2

A

ii

E o~

0

o

0 0

I

I

I

I

I

2

:3

4

5

hours Fig. 3. Effect of X-537A on Ca uptake (relative radioactivity) in kidney cells. The conditions are the same as those described in Fig. 2. X-537A (10 gg/ml) was added at min 55. The values are the mean of 4 experiments

Table 2. Effects of A23187 and X-537A on the total calcium of kidney cells Control

Ionophore

% change

P

11.0 _+0.51 (15) 1.01 _+0.05

7.37+_0.72 (16) 0.68_+0.07

-33%

<0.001

-33%

< 0.001

15.2 _ + 0 . 6 7 (27) 1.39_+0.06

9.78_+0.46 (23) 0.90_+0.04

-36%

<0.001

-36%

<0.001

A23187 1 gg/ml Cell calcium

(nmole/mg cell protein) (mmole/kg cell water)" X-537A 10 jxg/ml Cell calcium

(nmole/mg cell protein) (mmole/kg cell water)

The values are the mean+sE. The numbers in parenthesis indicate the number of determinations. a To convert nmole Ca/mg cell protein to mmole/kg cell water, the value has to be divided by 10.9 (The water content of LLC-MK2 kidney cells is 10.9 _+0.39 mg/mg protein; from Borle, 1970.)

58

A.B. Borle and R. Studer

2 A25187

0

I

0.1 !Jg/ml I

I

5 E v

=

2

. ~L__L -

----r--+ ~"

(D C:l

A23187

S_ 0 E --(,.3

2

o

I

I

X-537A

0

1.0 p g / r n l I

I

I0 # g / m l

I

I

I

I

2

3

hours Fig. 4. Effects of A23187 and of X-537A added at time 0 of isotope uptake on the relative radioactivity of kidney cells. Each graph represents the mean of 2 experiments

When the ionophore and the tracer are added together at time 0 of the labeling period, 45Ca uptake is markedly depressed as shown in Fig. 4. Under normal conditions, the curves of 45Ca uptake by isolated cells comprise two kinetic phases which can be easily separated by graphical analysis (Borle, 1970, 1975) or by nonlinear least square analysis. The fast compartment of exchange has a time constant of 1 to 2 min and represents extracellular Ca binding to membrane sites or to ligands of the glycocalyx. The curves of Ca uptake shown in Fig. 4 match the first kinetic component of a normal uptake curve (Borle, 1970, 1975). This would indicate that ionophores have little or no influence on the fast component of Ca exchange in isolated cells. This view is further supported by the fact that the level of the cells relative radioactivity observed after the addition of A23187 at min 150 (Fig. 2) is close to that obtained when one adds the ionophores at time 0 (Fig. 4). Fig. 2A shows that the relative radioactivity of the cells drops 60% when l gg A23187/ml of m e d i u m is added after 150 rain of uptake. Fig. 2b shows that increasing the concentration of A23187 to 5 gg/ml does not enhance significantly the fall in the relative radioactivity of the cells. This also supports the view that this ionophore does not affect

Ca Ionophores on Cellular Calcium

59

A23187 1.0 pg/rnl

o

t

20

"6 E

.oo..-~

15 13) .-i--

C~ :::3

I0

E E]

Vj EI

0

L

~

I

I

I

0

I

2

3

4

hours Fig. 5. Effect of A23187 (1 gg/ml) on Ca uptake by kidney cells incubated at different extracellular Ca concentrations. 9 = 1 mM Ca, 1 mM phosphate. 9 = 5 mM Ca, 0.2 mM phosp h a t e . . = 10 mN Ca, 0.2 mM phosphate. The phosphate was reduced to 0.2 mM to prevent precipitation of Ca phosphate. The ionophore was added at rain 155. The values are the mean of 6 experiments at 1 m~ Ca and the mean of 4 experiments at 5 and 10 mM Ca. The vertical bars represent the SEM

the first p h a s e o f Ca uptake. On the o t h e r h a n d , lowering the i o n o p h o r e c o n c e n t r a t i o n decreases its effect. F r o m 0.01 to 1.0 lag/ml, the % d r o p is closely related to the log o f the i o n o p h o r e c o n c e n t r a t i o n . On a weight basis, X - 5 3 7 A is 100 times less active t h a n A23187. T h e 4 2 % d r o p o b t a i n e d with 10 gg X - 5 3 7 A ml is close to the 37% fall o b s e r v e d with 0.1 ~tg A23187/ml.

Effect of extracellular calcium on the action of A23187. Since k i d n e y cells e x p o s e d to 1 ~g A23187/ml lose 3 3 % o f their total calcium a n d 6 0 % o f their r a d i o a c t i v i t y w h e n the extracellular calcium is 1.0 raM, we studied the m e d i u m Ca c o n c e n t r a t i o n at which the cells w o u l d actually gain Ca a n d a c c u m u l a t e m o r e tracer. Fig. 5 a n d T a b l e 3 s h o w t h a t with a m e d i u m Ca o f 5 raM, the cells' relative r a d i o a c t i v i t y still falls i m m e diately after the a d d i t o n o f the i o n o p h o r e , a l t h o u g h the ceils r e - a c c u m u -

60

A.B. Borle and R. Studer

Table 3. Effect of the ionophore A23187 on the total cell Ca of cultured kidney cells Medium Ca

Control

A23187 (1 ~tg/ml) % change

P

nmole/mg protein b 1.3 mN

11.0--+0.51 (15)

7.37-+0.72 (16)

--33%

<0.001

5.0 mMa

22.4--+ 1.35 (20) 33.9-+ 1.17 (24)

24.8 -+ 1.2 (19) 41.2 + 1.55 (19)

+ 10%

NS

+22%

<0.00I

10.0 ms a

Values are the mean_+s~. Numbers in parenthesis are the number of determinations. a At 5 and 10 mM Ca, the phosphate concentration was reduced to 0.2 mM to prevent the precipitation of Ca phosphate. b TO convert nmole Ca/mg cell protein to mmole/kg cell water the value has to be divided by 10.9. (The water content of LLC MK2 kidney cells is 10.9 + 0.39 mg/mg protein; ,from Borle, 1970.)

late the t r a c e r d u r i n g the next 80 min. H o w e v e r the total cell Ca does n o t increase significantly. A t a m e d i u m C a c o n c e n t r a t i o n o f 10 mM, A23187 first causes a small t r a n s i e n t fall in relative r a d i o a c t i v i t y , t h e n it significantly increases the rate o f 45Ca u p t a k e ; the total cell C a rises 22%.

Effect of Calcium ionophores on calcium efflux. 45Ca d e s a t u r a t i o n e x p e r i m e n t s were p e r f o r m e d at t h r e e different Ca c o n c e n t r a t i o n s : 0, 1.0 a n d 10 m s . Fig. 6 shows the effect o f 1 ~tg A 2 3 1 8 7 / m l o n the efflux rate coefficient (ERC) in five e x p e r i m e n t s . It is evident t h a t the i o n o p h o r e stimulates Ca efflux w h e t h e r or n o t Ca is present in the extracellular m e d i u m a n d even at high m e d i u m Ca c o n c e n t r a t i o n s . Fig. 7 presents the effects o f 10 ~tg X - 5 3 7 A / m l in the p r e s e n c e (1 mM) a n d in the absence o f C a in the m e d i u m . X - 5 3 7 A stimulates 45Ca efflux in b o t h conditions. Effects of ionophores on mitochondrial calcium. W e h a v e d e m o n s t r a t e d t h a t with an extracellular Ca c o n c e n t r a t i o n o f 1 mM, c u l t u r e d k i d n e y cells lose C a w h e n e x p o s e d to the i o n o p h o r e A23187. Since a large fraction of intracellular Ca is s e q u e s t e r e d in m i t o c h o n d r i a , their Ca c o n t e n t a n d their r a d i o a c t i v i t y s h o u l d also fall after A23187 a d m i n i s t r a tion. W e r e p e a t e d the e x p e r i m e n t s s h o w n in Fig. 2, in which the ionop h o r e is a d d e d 150 min after the beginning o f ~SCa u p t a k e w h e n the

Ca Ionophores on Cellular Calcium

61

A23187

250 20C -6 E

150

"6

I00

E~D o

50

0 (.9

0._

150 "6

I00

Ca,, = 0 o.,,., I

~

I

=lmM

"6 0

50

I

I

150 X

I

= I0 mM

I00 5O I

I

I

I

2

3

hours Fig. 6. Effect of A23187 (1 ~tg/ml) on the Ca efflux rate coefficient of kidney cells. The ionophore was added at rain 40 and removed at rain 90. The experiments were performed at different extracellular Ca concentrations: Ca=l mM (n=3); Ca=0 mM (n=2); Ca= 10 mu (n= 1)

cells are near their isotopic equilibrium. At that time the control cells received only the solvent, ethanol without ionophore, and the experimental group received 1 gg A23187/ml of suspending medium. The cells were homogenized l0 min later according to the technique described in the method section. Table 4 presents the results of this series of experiments. Within 110 min, the ionophore depressed the total cell calcium 20% and the cell radioactivity 43%. The total Ca and the radioactivity of the mitochondria were also depressed 25% and 39%, respectively.

62

A.B. Borle and R. Studer

X-557 A -6 'E

i

2OO

O O

"S

150

,,4--t--

r

c,

I00

,

J

~

=0

O O_

v E

50

O

",-7--

"~

150

O ~O

I00 X

=

50

~ ,I

I

=lmM

I

I

2

3

hours Fig. 7. Effect of X-537A (10 btg/ml) on the Ca efflux rate coefficient of kidney cells. Experimental conditions identical to those of Fig. 6

The drop in total mitochondrial Ca did not reach statistical significance probably because the determination of Ca in isolated mitochondria contains a large error, but the fall in radioactivity is significant. In control cells, the mitochondrial Ca can account for 33% of the total cell Ca. In the treated cells, 31% of the cell Ca is found in mitochondria. While the cells lost 2.6 nmoles Ca/mg cell protein after A23187 addition, the mitochondria lost 1.06 nmole Ca/mg cell protein. In other words, 41% of the total cell Ca loss is coming from the mitochondria.

Cells Freshly Isolated from Rat Kidney To make sure that the loss in cell Ca produced by the ionophores in LLC MK2 cells was not due to an aberrant property of cultured cells, we studied the effect of A23l 87 on the Ca content and the exchangeable pools of cells freshly isolated from animal tissues. The Ca uptake by freshly isolated kidney cells is shown in Fig. 8. The isotopic equilib-

Ca Ionophores on Cellular Calcium

63

Table 4. Effect of A23187 on the total Ca and on the relative radioactivity of cultured kidney cells and of their mitochondria Control

A23187 (1 lag/ml)

% change P

13.0 +_1.04 (15)

10.4 + 0 . 8 5 (16)

-20%

<0.05

(mmole/kg cell water)

1.19_+0.1

0.95_+0.08

Relative radioactivity (nmole/mg cell protein)

3.85_+0.19 (15)

2.21_+0.16 (16)

-43%

<0.001

85.5 _+17.5 (7)

64.4_+10.4 (7)

-25%

NS

(nmole/mg cell protein)

4.28+_0.88

3.22_+52

Relative radioactivity (nmole/mg cell protein)

1.38_+0.22 (7)

0.84_+0.19 (7)

-39%

<0.01

Cells Total calcium (nmole/mg cell protein)

Mitochondria Total calcium (nmole/mg mito protein)

Values are the mean_+sE. Numbers in parenthesis indicate the number of determinations.

rium is reached at 90 min and the relative radioactivity remains constant until 4 hr (not shown). After the addition of 1 Ixg A23187/ml, the cells relative radioactivity drops 22% (Fig. 8 and Table 5). Table 5 also shows that, after the addition of the ionophore, the total cell Ca concentration drops 17%. Thus, these results agree with those obtained in cultured kidney cells.

Kidney and Liver Slices Calcium Uptake Figs. 9 and 10 present the Ca uptake measured in kidney and liver slices. In both cases, isotopic equilibrium is reached after 60 rain and the relative radioactivity remains constant for the next three hr (not shown). After the addition of 5 gg A23187/ml medium, Ca uptake immediately increases. In kidney slices, the relative radioactivity reaches a new higher steady state after 60 rain (Fig. 9), whereas the radioactivity constantly rises in liver slices (Fig. 10). Table 6 shows that the average rise in Ca uptake during the two hr following administration of A23187

64

A.B. Borle and R. Studer

o_

12

Rot kidney cells

I0 E

ill ITI [

E

4

-6

2

I

A 23f87

pg/ml

0 )

I

L

I

l

I

2

3

4

hours Fig. 8. Ca uptake in ceils freshly isolated from rat kidneys. 45Ca was added at time 0. A23187 (1 gg/ml) was added at min 175. Each point is the mean _+sE of 4 experiments

Table 5. Effect of A23187 1 Bg/ml on the total Ca and 45Ca uptake in cells freshly isolated from rat kidney Control

A23187

% change P

Total cell calcium (nmole/mg protein) (mmole/kg cell water)

15.2 4-0.9a (13) 1.17+0.07

12.7 +0.3 b (18) 0.98+0.02

--17%

<0.001

-17%

<0.001

Calcium uptake (nmole/mg protein)

7.24_+0.5~ (20)

5.66+__0.3 a (16)

-22%

<0.001

a b a

Values from Valuesfrom Values from Values from

140 to 180 to 130 to 180 to

170 min. 230 rain. 170 min. 220 min.

is 2 7 % in k i d n e y slices a n d 4 4 % in liver slices. A t the s a m e time, the t o t a l C a c o n c e n t r a t i o n o f the slices also increases 3 3 % in k i d n e y a n d 3 6 % in liver. A few e x p e r i m e n t s were p e r f o r m e d in slices with 1 g g / m l of A23187. W i t h this c o n c e n t r a t i o n of i o n o p h o r e , no significant c h a n g e was observed, a n d the relative r a d i o a c t i v i t y r e m a i n e d c o n s t a n t f r o m 60 to 250 min.

Calcium content and radioactivity of mitochondria isolated from rat kidney and liver slices. W e h a v e s h o w n a b o v e t h a t A 2 3 1 8 7 decreases

Ca lonophores on Cellular Calcium

15

kidney

65

+,i

slices

-o

-6 E

IO

ID

5 pg/ml

A23187

5

E

OI

l

I

I

I

I

2

3

4

hours Fig. 9. Ca uptake in rat kidney slices. 45Ca was added at time 0. A23187 (5 gg/ml) was added at min 126. Each point is the mean_+sE of 4 experiments

tiver

r

Cr~

slices

30

-6 E

2C -I.-

E

IC A23187

5 pg/rnl

CJ 0 C

I

I

I

I

I

2

5

4

hours Fig. 10. Ca uptake in rat liver slices. 45Ca was added at time 0. A23187 (5 pg/ml) was added at rain 126. Each point is the mean+sE of 4 experiments

66

A.B. Borle and R. Studer

Table 6. Effect of A23187 (5 ~tg/ml) on the total Ca and the relative 4SCa radioactivity of rat kidney and liver slices Control

A23187

% change P

Total slice calcium (nmole/mg dry wt) (mmole/kg wet wOe (nmole/mg protein)

13.5 +_0.4 (28) 2.25 • 0.07 16.54+_0.5

17.9 +_0.4 (30) 2.99 _+0.7 21.9 _+0.5

+33%

<0.001

Relative radioaetivity (nmole/mg dry wt)

ll.6 • (28)

a

14.6 _+0.2b (30)

+27%

<0.001

Total slice calcium (nmole/mg dry wt) (mmole/kg wet wOe (nrnole/mg protein)

18.0 +-0.7 (23) 3.26 +_0.13 18.0 +-0.7

24.5 +- 1.3 (32) 4.44 • 0.24 24.5 +-1.33

+36%

<0.001

Relative radioactivity (nmole/mg dry wt)

17.5 +_0.5c (24)

25.2 _+0.8d (32)

+44%

<0.001

Kidney slices

Liver slices

Values are the mean +-sE. Numbers in parenthesis indicate the number of determinations Mean value measured between 35 and 125 rain. b Mean value measured between 170 and 260 min. ~ Mean value measured between 40 and 125 min. d Mean value measured between 140 and 260 rain. Kidney and liver slices contain 83.3_+0.25 and 81.9+_0.44% water, respectively. To convert nmole Ca/mg dry wt to nmole Ca/mg wet wt the values should be multiplied by 0.167 for kidney and by 0.181 for liver slices.

the t o t a l C a o f c u l t u r e d k i d n e y cells a n d t h a t this loss o f C a was reflected in a d r o p in the t o t a l Ca c o n c e n t r a t i o n a n d o f the r a d i o a c t i v i t y o f their m i t o c h o n d r i a . W e p o s t u l a t e d t h a t the C a c o n c e n t r a t i o n a n d the r a d i o a c t i v i t y o f m i t o c h o n d r i a isolated f r o m k i d n e y a n d liver slices c o u l d give s o m e i n f o r m a t i o n as to w h e t h e r the rise in Ca u p t a k e o f slices o b s e r v e d after A23187 a d m i n i s t r a t i o n is actually due to a shift o f C a into the cell. I f this were so, the t o t a l Ca a n d the relative activity o f m i t o c h o n d r i a s h o u l d also increase. T a b l e 7 shows t h a t this is n o t the case, at least in rat kidney. I n d e e d , despite the 3 3 % rise in the t o t a l Ca o f k i d n e y slices a n d the 2 7 % rise in their r a d i o a c t i v i t y o b s e r v e d on the a d d i t i o n o f A23187 ( T a b l e 6), the m i t o c h o n d r i a isolated f r o m the same tissue, t r e a t e d in an identical fashion, s h o w a 3 6 % fall in t o t a l Ca a n d a 3 2 % d r o p in r a d i o a c t i v i t y ( T a b l e 7). T h e s e results strongly suggest t h a t A23187 does n o t increase the cellular a c c u m u l a t i o n o f Ca

Ca Ionophores on Cellular Calcium

67

Table 7. Total Ca and relative radioactivity of mitochondria isolated from control kidney slices and slices exposed to A23187 (5 gg/ml) for 10 min Control

A23187

% change P

Kidney mitochondria Total calcium (nmole/mg mito protein) (nmole/mg slice protein)

27.2 +7.7 (12) 9.52 + 2.7

17.4 -+3.5 (12) 6.09 +. 1.23

-36%

0.05"

Relative activity (nmole/mg mito protein)

6.64-+0.8 (16)

4.52_+0.5 (16)

-32%

0.001 a

Liver mitochondria Total calcium (nmole/mg mito protein) (nmole/mg slice protein)

24.8 -+2.5 (I I) 4.98 _+0.5

27.6 _+2.7 (11) 5.55 -+0.54

+11%

NS

Relative activity (mnole/mg mito protein)

16.2 -+3.0 (11)

14.3 -+2.8 (12)

-12%

NS

a

Values are the mean-+sm Numbers in parenthesis are the number of determinations. Correlated t test.

and of tracer in the cells. On the contrary, the cells seem to lose both in spite of the Ca accumulation produced by the ionophore in whole slices. The relative radioactivity of kidney slices at isotopic equilibrium is 37% larger than that of freshly isolated cells (Tables 5 and 6). Since the relative radioactivity is a measure of the exchangeable Ca pools, this suggests that 37 % of the exchangeable Ca of kidney slices is extracellular. It is possible, therefore, that the net accumulation of Ca and of tracer observed in kidney slices after the addition of A23187 occurs in the extracellular interstitium and not in the intracellular compartment. The results obtained with mitochondria isolated from liver slices are not as clear. Table 7 shows that the total mitochondrial Ca and their radioactivity are essentially unchanged. The small rise in total Ca and the small decrease in radioactivity are not statistically significant. Nevertheless, in light of the large rise in the total Ca and in the radioactivity of the liver slices ( + 36% and + 4 4 % , respectively, as shown in Table 6), one would have expected the ionophore to increase the mitochondrial concentration of both, if these changes had occured in the cells. Comparison between Tables 6 and 7 also shows that, in control kidney slices, 57% of the Ca is found in mitochondria. In liver, the mitochondrial Ca accounts for 28% of the total slice Ca.

68

A.B. Borle and R. Studer Discussion

The Ca ionophore A23187 is frequently used as a tool to study cellular functions which are believed to be regulated by cytosolic Ca. Several investigators have reported that this ionophore increases the uptake of 45Ca in a variety of tissues (Reed & Lardy, 1972a; Prince et al., 1973; Massini & Luscher, 1974; Dziak & Stern, 1975). It is also frequently observed that A23187 fails to elicit a cellular response in the absence of extracellular Ca (Prince et al., 1973; Russel, Hansen & Thorn, 1974; Williams & Lee, 1974; Clyman et al., 1975; Garcia, Kirpekar & Prat, 1975; Cochrane et al., 1975). F r o m such observations, many assume that A23187 produces a net shift of Ca from the extracellular milieu to the cell, increases the cytosolic Ca, and thereby triggers the process which is dependent on a rise in cytosolic Ca activity. Although such a sequence of events could very well occur, it is far from being proven by the available data. On the other hand, several reports show that A23187 can induce activation of sea urchin eggs, myogenic cell fusion, and insulin release from pancreatic islets in the absence of extracellular Ca (Schudt & Pette, 1975; Chambers et al., 1974; Karl et al., 1975). In these papers, the authors propose that A23187 mobilizes Ca from intracellular compartments. The results presented in this paper strongly support the latter hypothesis. Indeed we have conclusively shown that A23187 decreases the total Ca and the radioactivity of cells and of their mitochondria. Our results also cast doubt on the validity of the argument that an increased 45Ca accumulation by a tissue is evidence of a shift of Ca into the cells. It is indeed difficult to defend the postulate that an increased accumulation of Ca by the cells could coexist with a decreased Ca content and a fall in the accumulated radioactivity of their mitochondria. Admittedly Ca ionophores increase the permeability of the cell plasma membrane and of the mitochondrial membrane to Ca (Reed & Lardy, 1972b; Pressman, 1973; Sordhal, 1974; Binet & Volfin, 1975). Although the concentration of cytosolic-free Ca has never been measured, some evidence supports the view that it increases after the addition of Ca ionophores. (Rose & Lowenstein, 1976; Desmedt & Hainaut, 1976). Our results obtained with red blood cells only confirm what was expected; cellular Ca uptake is dramatically increased and cell Ca concentration rises in the presence of A23187. Erythrocytes, however, may not be a good model for studying the effects of Ca ionophores because they lack mitochondria. In other cells one may wonder, first, whether these

Ca Ionophores on Cellular Calcium

69

ionophores ever reach the mitochondrial membranes and, if they do, whether the release of Ca from mitochondria significantly contributes to the rise in cytosolic Ca. Second, if Ca influx into the cell and Ca release from mitochondria both occur at the same time, what will be the net effect on the total cell Ca? If the stimulation of Ca transport across the plasma membrane predominates, the cells would presumably gain Ca. On the other hand, if the release of Ca from mitochondria is the most important effect of Ca ionophores, the cells may or may not lose Ca depending on the driving forces determining Ca movements. The results presented here suggest that Ca ionophores do penetrate the cell membrane, reach the mitochondria, and stimulate Ca release from this intracellular compartment. We cannot exclude, of course, that other unidentified compartments of kidney cells are capable of sequestering Ca and of releasing it under the influence of ionophores. An alternative explanation, yet completely unproven, could be that the high cytosolic Ca caused by an increase in Ca influx across the plasma membrane could increase the permeability of the mitochondrial membranes to Ca and release the Ca sequestered in the mitochondria. An argument against such an explanation is the fact that both A23187 and X-537A stimulate Ca efflux from the cells in the absence of extracellular Ca (Figs. 6B and 7B). In these experiments, the cytosolic Ca activity obviously could not have been raised by an increased Ca influx. Fig. 6 C also shows that a high extracellular Ca concentration does not prevent the increased Ca efflux triggered by A23187. Whatever the mechanism, it is clear that both the mitochondria and the cells lose Ca after the addition of ionophores. The driving force causing the net shift of Ca from the cell to the extracellular fluid is unknown. The membrane potential of the kidney cells used in these experiments (LLC-MK2) is - 1 2 . 3 mV (Redmann, 1971), the inside of the cell being negative. The cytosolic Ca activity has never been measured after the addition of ionophores but there is no reason to believe that it exceeds the extracellular Ca activity. Thus, even if the ionophores depress the membrane potential to zero, we still have to explain the 30% loss of total cell Ca. One could speculate that, despite the increased plasma membrane permeability to Ca, the active transport of Ca out of the cell is maximally stimulated by the high cytosolic Ca activity and can extrude in less than 10 rain 30% of the total cell Ca released from the mitochondria and possibly from other intracellular compartments. Otherwise, one has to postulate that an intracellular compartment capable of sequestering more than 30% of the cell Ca has a free Ca concentration exceeding 1 mM. In

70

A.B. Borle and R. Studer

fact, it should be significantly higher than 1 mM since, at a medium calcium concentration of 5 mM, the cells fail to gain Ca after the addition of A23187. These results are not limited to cultured kidney cells. It has been reported that A23187 enhances the release of Ca from heart and skeletal muscle (Holland et al., 1975; Schudt & Pette, 1975). The ionophores A23187 and X-537A induce parthenogenesis in sea urchin eggs, a process regulated by intracellular Ca, in the absence of external divalent cations (Chambers et al., 1974). Finally, the stimulation of insulin release from pancreatic islets produced by A23187 has been shown to occur in the absence of extracellular Ca (Karl et al., 1975). Our own data show that cells freshly isolated from rat kidney lose Ca after administration of A23187. Furthermore, the mitochondria isolated from kidney slices incubated with 45Ca lose 20% of their Ca content and radioactivity after administration of A23187. Presently, we have no substantiated explanation for the conflicting findings showing that A23187 increases Ca uptake by slices or by whole tissues while it decreases the Ca concentration of ceils and of their mitochondria. One could postulate, however, that this ionophore increases the accumulation of Ca in the extracellular interstitium of the tissues. Hyono et al. (1975) have presented evidence that A23187 at low concentrations can bind two Ca ions per molecule and this ratio changes at higher ionophore concentrations to one Ca ion per two ionophore molecules. Thus, it could be theoretically possible for A23187 to attach with one binding site to the Ca bound to some ligands of the extracellular interstitium and, with the other binding site, trap in this interstititium a significant a m o u n t of Ca labelled and unlabelled from the incubating medium. Perhaps it may be relevant to note that higher concentrations of ionophore (5 ~tg/ml) must be used to produce an effect in tissue slices than that used in isolated cells, where 0.1 gg/ml produce significant Ca shifts. This could indicate that an important fraction of A23187 might be bound to some ligands of the interstitium. If one postulates that ionophores increase the cytosolic Ca activity by mobilizing Ca from an intracellular compartment, the lack of action of A23187 and of other Ca ionophores in the absence of extracellular Ca remains unexplained, Again we can only speculate that a tissue incubated in Ca-free media will lose a significant fraction of its intracellular Ca. Indeed, experiments to be published (Borle, in preparation) show that kidney cells incubated in a Ca-free medium lose 80% of their total Ca within an hour. Since the largest fraction of the intracellular Ca

Ca Ionophores on Cellular Calcium

71

is found in mitochondria, it is evident that incubation in Ca-free media will severely deplete these internal stores of Ca. In these conditions, ionophores may not be capable of increasing cytosolic Ca by mobilizing it from depleted intracellular stores. In some cases, if the cells are not totally depleted, ionophores may still possibly increase the cytosolic Ca in the absence of extracellular Ca (Schudt & Pette, 1975; Chambers et al., 1974; Karl et al., 1975). In conclusion, we believe that the increased 45Ca uptake by a tissue exposed to A23187 and the lack of effect of this ionophore in Ca-free media, cannot be taken as a proof that this carboxylic antibiotic acts by producing a net shift of Ca from the extracellular fluids to the cytosol. Such results could still be consistent with the idea that ionophores increase the cytosolic Ca by mobilizing Ca from subcellular compartments (Rose & Lowenstein, 1976). Our results support the view that Ca ionophores increase the permeability of plasma and mitochondrial membranes to Ca and that they increase the cytosolic Ca concentration. However, the increased cytosolic free Ca is not due to an net shift of Ca from the extracellular fluids into the cell. On the contrary, it is clear that these divalent cation ionophores produce a net loss of cell Ca and that the source of the increased cytosolic Ca must be intracellular, presumably the mitochondria. This work was supported by U.S. Public Health Service grant NAM-07867 from the National Institutes of Health. The technical assistance of Mrs. Mary Wusylko and Mrs. Sandra Gregor is gratefully acknowledged. The ionophore A23187 was a generous gift from Dr. Robert Hamill, Lilly Research Laboratories and ionophore X-537A was from Dr. W.E. Scott, Hoffmann-La Roche, Inc. Both gifts are gratefully acknowledged.

References Binet, A., Volfin, P. 1975. Effect of the A23187 ionophore on mitochondrial membrane Mg + + and Ca + +. FEBS Lett. 49:400 Borle, A.B. 1970. Kinetic analyses of calcium movements in cell cultures. III. Effects of calcium and parathyroid hormone in kidney cells. J. Gen. Physiol. 55:163 BorIe, A.B. 1975. Methods for assessing hormone effects on calcium fluxes in vitro. In: Methods in Enzymology. Hormone Action. Vol. 39, part D, p. 513. J.G. Hardman and B.W. O'Malley, editors. Academic Press, New York Borle, A.B., Briggs, F.N. 1968. Microdetermination of calcium in biological materials by automatic fluorometric titration. Anal. Chem. 40:339 Chambers, E.L., Pressman, B.C., Rose, B. 1974. The activation of sea urchin eggs by the divalent ionphores A23187 and X-537A. Biochem. Biophys. Res. Commun. 60:126 Clyman, R.I., Blacksin, A.S., Syndler, Y.A., Manganiello, V.C., Vaugh, M. 1975. The role of calcium in regulation of cyclic nucleotide content in human umbilical artery. J. Biol. Chem. 250:4718

72

A.B. Borle and R. Studer: Ca Ionophores on Cellular Calcium

Cochrane, D.E., Douglas, W.W., Mouri, T., Nakazato, Y. 1975. Calcium and stimulussecretion coupling in the adrenal medulla: Contrasting stimulating effect of the ionophores X-537A and A23187 on catecholamine output. J. Physiol. (London) 252:363 Desmedt, J.E., Hainaut, K. 1976. The effect of A23187 ionophore on calcium movements and contraction powers in single barnacle muscle fibers. J. Physiol. (London) 257:87 Dziak, R., Stern, P. 1975. Parathyromimetic effects of the ionophore A23187 on bone cells and organ cultures. Biochem. Biophys. Res. Commun. 65:1343 Garcia, A.G., Kirpekar, S.M., Prat, J.C. 1975. A calcium ionophore stimulating the secretion of catecholamines from the cat adrenal. J. Physiol. (London) 244:253 Holland, D.R., Armstrong, W.McD., Steinberg, M.I. 1975. 4~Ca fluxes induced by A23187 in guinea pig left atria. Physiologist 18:251 Hyono, A., Hendriks, T., Daemen, F.J.M., Bonting, S.L. 1975. Movement of calcium through artificial lipid membranes and the effects of ionophores. Biochim. Biophys. Acta 389: 34 Karl, R.C., Zawalich, W.S., Ferrendelli, J.A., Matchinsky, F.M. 1975. The role of Ca ++ and cyclic adenosine 3':5'-monophosphate in insulin release induced in vitro by the divalent cation ionophore A23187. J. Biol. Chem. 250:4575 Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the folin phenol reagent. Y. Biol. Chem. 193:265 Massini, P., Luscher, E.F. 1974. Some effects of ionophores for divalent cations on blood platelets. Comparison with the effects of thrombin. Biochim. Biophys. Acta 372:109 Pressman, B.C. 1973. Properties of ionophores with broad range cation selectivity. Fed. Proc. 32:689 Prince, W.T., Rasmussen, H., Berridge, M.J. 1973. The role of calcium in fly salivary gland secretion analyzed with the ionophore A23187. Biochim. Biophys. Acta 329:98 Redman, K. 1971. Measurement of electric membrane potentials of cultivated single cells by means of microelectrodes. Acta Biol. Med. Ger. 27:55 Reed, P.W., Lardy, H.A. 1972a. Antibiotic A23187 as a probe for the study of calcium and magnesium function in biological systems. In : The Role of Membranes in Metabolic Regulation. p. III. M.A. Mehlman and R.W. Hauson, editors. Academic Press, New York Reed, P.W., Lardy, H.A. 1972b. A23187: A divalent cation ionophore. J. Biol. Chem. 247: 6970 Rose, B., Loewenstein, W.R. 1976. Permeability of a cell junction and the local cytoplasmic free ionized calcium concentration: A study with aequorin. J. Membrane Biol. 28:87 Russel, J.T., Hansen, E.L., Thorn, N.A. 1974. Calcium and stimulus-secretion coupling in the neurohypophysis. III. Ca 2+ ionophore (A23187) induced release of vasopressin from isolated rat neurohypophyses. Acta Endocrinol. 77:443 Schudt, C., Pette, D. 1975. Influence of the ionophore A23187 on myogenic cell fusion. FEBS Lett. 59: 36 Shain, S.A. 1972. In vitro metabolism of 25-hydroxycholecalciferol by chick intestinal and renal cell preparations. Identification of metabolic product as 1,25-dihydroxycholecaliciferol and delineation of its metabolic fate in intestinal cells. J. Biol. Chem. 247:4393 Sordhal, L.A. 1974. Effect of magnesium, ruthenium red, and the antibiotic ionophore A23187 on initial rates of calcium uptake and release by heart mitochondria. Arch. Biochem. Biophys. 167:104 Williams, J.A., Lee, M. 1974. Pancreatic acinar cells: Use of a Ca + + ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling. Biochem. Biophys. Res. Commun. 60" 542